Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web (Atrax and Hadronyche) spider venoms

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve-muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5110) produced muscle contracture and fasciculation when applied to the nerve-muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders. (C) 2001 Elsevier Science Ltd. All rights reserved.

Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Life history parameters and biocontrol potential of the mealybug parasitoid Coccidoxenoides peregrinus (Timberlake) (Hymenoptera: Encyrtidae): Asexuality, fecundity and ovipositional patterns

Properties relevant to the ovipositional activity and lifetime productivity of Coccidoxenoides peregrinus (Timberlake) were assessed in the laboratory, to determine the potential of this species as a biocontrol agent against the citrus mealybug, Planococcus citri (Risso). In general, this species has not performed well in orchards, except for a few localities on different continents. The mode of reproduction of C peregrinus is almost entirely thelytokous, with males produced sporadically and at low frequency. The females have both pro-ovigenic and synovigenic traits, which raises questions of the utility of this distinction. The females have a high reproductive potential with 10-20 eggs per day available within the first two days (after a short (12 h) pre-oviposition period), and 80-150 eggs per day thereafter until death at about eight days. Mean lifetime fecundity was 239.2 +/- 34.3 eggs. C peregrinus oviposits across a range of P. citri instars, but productivity relies predominantly on second instar hosts. Second stage (N2) hosts received most eggs in choice (about 52%) and no-choice (about 50%) tests. Most eggs deposited into N2 hosts (82%) reached adult stage whereas only a few of those deposited into N1 and N3 (about 5% each) developed successfully. The haemolymph of parasitised reproductive mealybugs contained granular structures and no parasitoid eggs were found 24 h after exposure to ovipositing wasps. Also, no wasps emerged from parasitised adult hosts that were kept alive. Parasitoid eggs deposited into adult hosts were presumed encapsulated and destroyed, as control mealybugs (not exposed to female wasps) had no granular structures in their haemolymph. Wasps exposed to an abundance of hosts soon started ovipositing, but only for a relatively short time each day (about 2.5 h out of a 7 h exposure). They stopped ovipositing despite eggs judged to be mature in their ovaries. The reproductive output of C peregrinus is discussed in relation to the ecological factors that could influence this output, and the implications for biocontrol are discussed. (C) 2003 Elsevier Inc. All rights reserved.

Cryptic false spider mites: a new genus, Austrolinus, and a review of the family Linotetranidae (Acari : Prostigmata : Tetranychoidea)

The Linotetranidae (Acari: Tetranychoidea) is a poorly known group of cryptic false spider mites associated with grasses and sedges. We review the family at the world level, provide the first phylogenetic analysis of the family, and describe the first Australian representatives: Austrolinus, gen. nov. and two new species: A. arenulus and A. kinnearae. Linotetranidae is redefined, and keys are provided for the families of the Tetranychoidea, and for all described genera and species of Linotetranidae.

Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response

During oviposition, the parasitoid wasp Cotesia congregata injects polydnavirus, venom, and parasitoid eggs into larvae of its lepidopteran host.. the tobacco hornworm, Manduca sexta. Polydnaviruses (PDVs) suppress the immune system of the host and allow the juvenile parasitoids to develop without being encapsulated by host hemocytes mobilized by the immune system. Previous work identified a gene in the Cotesia rubecula PDV (CrV1) that is responsible for depolymerization of actin in hemocytes of the host Pieris rapae during a narrow temporal window from 4 to 8 h post-parasitization. Its expression appears temporally correlated with hemocyte dysfunction. After this time, the hemocytes recover, and encapsulation is then inhibited by other mechanism(s). In contrast, in parasitized tobacco hornworm larvae this type of inactivation in hemocytes of parasitized M. sexta larvae leads to irreversible cellular disruption. We have characterized the temporal pattern of expression of the CrV1-homolog from the C. congregata PDV in host fat body and hemocytes using Northern blots, and localized the protein in host hemocytes with polyclonal antibodies to CrV1 protein produced in P. rapae in response to expression of the CrV1 protein. Host hemocytes stained with FITC-labeled phalloidin, which binds to filamentous actin, were used to observe hemocyte disruption in parasitized and virus-injected hosts and a comparison was made to hemocytes of nonparasitized control larvae. At 24 h post-parasitization host hemocytes were significantly altered compared to those of nonparasitized larvae. Hemocytes front newly parasitized hosts displayed blebbing, inhibition of spreading and adhesion, and overall cell disruption. A CrV1-homolog gene product was localized in host hemocytes using polyclonal CrV1 antibodies, suggesting that CrV1-like gene products of C. congregata's bracovirus are responsible for the impaired immune response of the host. (C) 2005 Elsevier Ltd. All rights reserved.

Why is Coccidoxenoides perminutus, a mealybug parasitoid, ineffective as a biocontrol agent - Inaccurate measures of parasitism or low adult survival?

Coccidoxenoides perminutus achieves only low levels of parasitism of its host Planococcus citri in southeast Queensland citrus. Two possible causes were investigated. Adult survival under natural conditions was assessed to determine whether providing adult food sources could enhance survival. Behavioural changes of hosts, induced by C perminutus parasitism, was also investigated to establish if parasitised P. citri move from their feeding site to seek protected shelters some distance away and are thus not accounted for in field assessments of parasitism rates. Unparasitised mealybugs placed in the field for two periods were retrieved before the effects of parasitism were manifested and parasitism rates were still low (0.3% at 5 days and 1.2% at 10 days). Levels of locomotion of P. citri exposed to C perminutus were compared with those of unexposed ones. Parasitised mealybugs, regardless of instar, undergo behavioural changes. In comparison to unparasitised controls, the mealybugs become highly active 7-14 days after exposure to wasps. All parasitised mealybugs undergo physical changes, their body becomes cylindrical, their legs go so rigid that the mealybugs become immobile, and this signifies the typical mummy appearance. All mealybugs that became mummies eventually fell from the host lemon fruit because of impaired locomotion and were caught on sticky traps that had been placed beneath the lemons. Consequently, their final site of mummification was not established. C perminutus adults provided with nectar or honey survived longer (about 5 days) in the field than those without food (about a day). Nectar from two plant species, Alpinia zerumbet and Datura candida, proved to be good sources of food for the adult wasps, and were comparable in quality to honey. The low level of parasitism achieved by C perminutus in southeast Queensland citrus thus appears to be a consequence of the short adult life and the negative effects of a harsh environment. Provision of a suitable food source (e.g., nectar) may well enhance levels of parasitism in the field. (c) 2005 Elsevier Inc. All rights reserved.

Refinement of a cell line based artificial diet for rearing the parasitoid wasp, Trichogramma pretiosum

An artificial diet incorporating insect cells originally developed for Trichogramma australicum Girault (Hymenoptera: Tricho-grammatidae) was successfully used to rear Trichogramm pretiosum Riley (Hymenoptera: Trichogrammatidae). To refine the diet, individual components were removed. Chicken egg yolk and the insect cells were identified as the most important components for T. pretiosum development. Their removal resulted in few pupae and no adults. Removal of Grace's insect medium, a common component of artificial diets, was found to markedly improve the development of T pretiosum, producing 60% larva to pupa transition and 19% pupa to adult transition. There was no significant difference in T pretiosum development on diets in which milk powder, malt powder or infant formula were interchanged, despite differences in nutrient composition. The use of yeast extract resulted in significantly higher survival to the adult stage when compared with yeast hydrolysate enzymatic and a combination of yeast extract and yeast hydrolysate enzymatic. Comparison of four antimicrobial agents showed the antibacterial agent Gentamycin and the antifungal agent Nystatin had the least detrimental effect on T pretiosum development. The use of insect cell line diets has the potential to simplify artificial diet production and significantly reduce T pretiosum production costs in Australia compared to diets using insect hemolymph or the use of natural or factitious hosts. (c) 2005 Elsevier Inc. All rights reserved.

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MITI, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pia ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1 mu M, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors. (c) 2005 Elsevier Inc. All rights reserved.

Oviposition and reproductive performance of a generalist parasitoid (Trichogramma pretiosum) exposed to host species that differ in their physical characteristics

The response of generalist egg parasitoids to alternative natural hosts that are present simultaneously is not well known. We investigated the behavior of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) in relation to two field hosts Helicoverpa armigera Hubner and Spodoptera litura Fabricius, in choice and no choice tests. We quantified the effects of natal host species and post-emergence adult age on the oviposition preference of the parasitoids. H. armigera eggs were consistently preferred over S. litura eggs, regardless of the natal host and adult age. When only S. litura eggs were available as hosts, they were parasitized at statistically similar rates to H. armigera eggs (average of 17 +/- 2.7 vs. 13 +/- 3.0, H. armigera to S. litura). The adult lifespan and lifetime fecundity of T. pretiosum were variable but were affected by natal host species and/or host species to which they were exposed. Mean lifespan and fecundity of parasitoids that had developed in H. armigera eggs and were exposed to H. armigera eggs for oviposition were 13.9 +/- 1.8 days and 98.7 +/- 11.0 adult offspring. By contrast, those that developed in S. litura eggs and were exposed to S. litura eggs for oviposition lived for 7 +/- 0.9 days and produced 53.8 +/- 8.0 adult offspring. The ovigeny index (OI) was significantly lower in the parasitoids exposed to H. armigera eggs than in those exposed to S. litura eggs, regardless of the natal host, indicating that H. armigera eggs sustain the adult parasitoids better than S. litura eggs. These results are used to predict parasitoid behavior in the field when both hosts are available. (c) 2006 Elsevier Inc. All rights reserved.